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Biocatalysis — key to sustainable industrial chemistry
Roland Wohlgemuth
The ongoing trends to process improvements, cost reductions and increasing quality, safety, health and environment requirements of industrial chemical transformations have strengthened the translation of global biocatalysis research work into industrial applications. One focus has been on biocatalytic single-step reactions with one or two substrates, the identiﬁcation of bottlenecks and molecular as well as engineering approaches to overcome these bottlenecks. Robust industrial procedures have been established along classes of biocatalytic single-step reactions. Multi-step reactions and multi-component reactions (MCRs) enable a bottom-up approach with biocatalytic reactions working together in one compartment and recations hindering each other within different compartments or steps. The understanding of the catalytic functions of known and new enzymes is key for the development of new sustainable chemical transformations.
Address Sigma–Aldrich, Industriestrasse 25, CH-9470 Buchs, Switzerland Corresponding author: Wohlgemuth, Roland (roland.wohlgemuth@sial.com)

omical, energy saving, and environment-friendly production procedures. The global needs for clean manufacturing technologies, nonrenewable raw materials, management of hazardous chemicals and waste present new research challenges to both chemistry and biotechnology. These sciences are taking up these challenges and the initiatives in Green/sustainable chemistry [2,3] and white/industrial biotechnology [4] have emerged in their disciplines independently. It is therefore of crucial importance for the success of implementation and translation of science and technology into standard industrial practice to develop a common chemistry–biotechnology interface. One common opportunity for improvement and invention is the current use of protecting groups for overcoming nonselective and incompatible reactivities in synthesis and biomimetic as well as enzyme-catalyzed synthesis can provide the selectivities needed to overcome barriers [5]. The manufacturing of molecular complexity from simple starting materials with a minimum number of steps, avoiding protection–deprotection loops and orientation towards function of the product attract much interest and biocatalytic process steps are well positioned for contributing to the solutions of the above-mentioned challenges [6]. The creation of sustainable value by viable industrial processes and synthetic pathways requires not only research progress in chemistry and biotechnology, but in addition the integration of research from molecular and engineering sciences, thereby enabling a large range of industrial biotransformations [7–10]. As reaction development serves different practical needs, progress in the working areas single-step reactions, multi-step reactions, and multi-component reactions (MCRs) will be discussed in the following sections. Despite the enormous achievements in the chemical synthesis of organic compounds, once believed to be accessible only by biological processes and ‘vital forces’, over the past two centuries, many present state-of-the-art processes are highly inefﬁcient [3]. This and additional boundary conditions like safety, health and environment issues in industrial processes have revitalized the interest in the discovery/invention of novel biocatalytic reactions and reaction methodologies, which have been evolved by nature to achieve highly efﬁcient and selective transformations. Therefore the section on the development of new biocatalytic reaction methodology addresses this important industrial innovation area.

Introduction
The creation of value-added products by chemical transformations has contributed signiﬁcantly to the quality of life over the centuries and has reached a high level, but it has been suggested that many of the stoichiometric reactions in current use should be replaced by catalytic processes [1]. Although catalytic tools are not only a cornerstone of our present economy and society, but also a key feature of basic life processes, most of the catalysts used in the automotive, fuel reﬁning, and chemical industries consist either of inorganic, organometallic or of organic catalysts in heterogeneous form, as for example, catalysts involved in pollutant removal from the exhaust leaving the car engines. The use of biocatalysts in chemical transformations has really taken off with the focus on safe, healthy, resource efﬁcient and econwww.sciencedirect.com

Industrial biocatalytic single-step reactions
The early success of single biocatalytic reaction steps in classical organic synthesis schemes has led to an
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increasing number of established industrial processes and continues to be a useful approach for the introduction of biocatalysis into industrial practice. The discovery and development of novel biocatalytic reaction steps can thereby focus on overcoming synthetic bottleneck reactions and improving the performance of existing chemical reactions according to industrial requirements. Biocatalytic versions of reactions which are impossible or impractical by existing chemistry tools generate high interest and stimulate further process research and development work in industry.

Oxidations and reductions catalyzed by oxidoreductases have progressed towards the tools of choice (Figure 1) due to their improved performance with respect to reaction selectivity, safety, health, and environment aspects. Selective introduction of one or two oxygen atoms by biocatalysts has continued to attract a lot of industrial interest. Among the reactions introducing one oxygen atom, selective asymmetric hydroxylations, epoxidations, and Baeyer–Villiger oxidations [11–13,14] have made signiﬁcant progress and are of interest for the oxyfunctionalization of inexpensive organic building blocks. Selective biocatalytic oxidations of one out of several hydroxygroups, as for example, in alcohols and sugars, continue to be of industrial interest since the thirties of the last century and have additional sustainability beneﬁts compared to the classical chemical oxidations [7]. Since classical chemical oxidations often use stoichiometric oxidants in excess, the selective removal of remaining oxidants is decisive for the product quality and enzymatic methods have become standard practice in production. Depending on the enzyme properties and the cofactor recycling system, both the oxidative and the reductive directions of an oxidoreductase application are of interest [15,16]. Sustainable enzymatic reductions of aldehydes and ketones are reliable, scalable and inexpensive routes to optically active alcohols and have been extensively employed in organic synthesis despite the vast number of asymmetric reductions [17]. Even in the area of the reduction of carbon–carbon double bonds, where catalytic hydrogenation with hydrogen gas in autoclaves is performed routinely, new asymmetric biocatalytic reductions of activated alkenes bearing an electron-withdrawing group have become interesting methods for preparing the corresponding saturated products in up to >99% ee and for side stepping the use of hydrogen gas [18]. High enantioselectivity was also observed for the asymmetric reduction of activated a,b-unsaturated enones catalyzed by pentaerythritol tetranitrate reductase for reaction product stereogenic centers at the beta-carbon atom [19]. Enoate reductases have also been used for the conversion of a series of a,b-unsaturated nitriles to the optically
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As in nature, industrial biocatalytic aminations have been performed by the two different routes of transamination and reductive amination. The use of amino acid dehydrogenases in reductive amination of prochiral precursors continues to play an important role in the enzymatic production of D-enantiomers and L-enantiomers of both natural and non-natural amino acids. Transaminases have obtained increased interest for the asymmetric synthesis of amines from prochiral ketones [21–24] and amination is becoming a key reaction (Figure 2) in industrial biotransformations [9]. New routes to nonchiral amines are also of interest and a new biocatalytic transamination of pyridoxal-50 -phosphate has been achieved with complete conversion [25]. The efﬁciency of the manufacturing process for the antidiabetic compound sitagliptin has been greatly improved by replacing the Rh(Jobiphos)catalyzed asymmetric hydrogenation of an enamine at high pressure with a direct transaminase-catalyzed amination of prositagliptin ketone [26]. The best engineered enzyme could convert 200 g/l prositagliptin ketone to sitagliptin with an excellent ee of >99.95%. A 53% productivity increase, 19% waste reduction, elimination of heavy metals, cost reductions and avoiding specialized high-pressure hydrogenation equipment have been found as speciﬁc advantages of the biocatalytic process [26].
Glycosylation reactions

As selective chemical glycosylation reactions require a substantial synthetic effort involving various protecting group chemistries in organic solvents, the use of glycosyltransferases for coupling glycosyl donors to nonprotected acceptors in aqueous media (Figure 3) continues to attract a lot of interest [27–29]. Methods based on the application of glycosyltransferases are currently recognized as being the most effective for the preparation of complex and highly pure oligosaccharides [30]. The trihexosylceramides Gb3 and iGb3 have been synthesized by speciﬁc galactosyltransferases using lactosylceramide as acceptor [31]. Sialyltransferases have been used in chemoenzymatic or whole-cell approaches for the synthesis of a large library of sialoside standards and derivatives [32]. Carbohydrate-based drug design makes use of various glycosyltransferases for the production of novel glycosylated compounds, as no single universal glycosyltransferase has been found [33]. The ﬁnal hexose to be transferred from the NDP-hexose to the aglycon can thereby be diversiﬁed by a variety of enzymes like dehydratases, epimerases and aminotransferases.
Hydrolysis and reverse hydrolysis reactions

On the basis of the vast number of established enzymatic reactions using hydrolases in aqueous and nonaqueous systems, this area has become well established and new
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Figure 1

Selected biocatalytic oxidation and reduction reactions. The cyclohexanone-monooxygenase-catalyzed Baeyer–Villiger oxidation of bicycloheptenone has been applied industrially by Sigma–Aldrich with the substrate-feed-product-recovery-technology (SFPR) using Optipore L-493 as adsorber for high space-time yield [14].

applications appearing in various ﬁelds of organic chemistry can build on this experience (Figure 4). The largescale availability of many hydrolases like acylases, amidases, esterases, lipases, proteases and their ease of use without any cofactors has been a key factor for the rapid growth of this reaction class in industry [34]. The robustness and scalability of these reactions with standard equipment have been useful for resolutions, deracemizations, desymmetrizations in early steps or mild
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deprotections in late steps of a synthesis. The complete conversion of a substrate into one product of high enantiomeric purity is particularly attractive, as for example, in desymmetrizations of prochiral diols or diesters. Inexpensive acyl donors like acids or simple esters are preferred for cost-sensitive productions, but require tools to drive reactions to completion. Lipase-catalyzed acylations with activated acyl donors like enol esters and acid anhydrides are practically irreversible. Lipase-catalyzed
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polymerization in an organic solvent or one bulk monomer is advantageous in reducing energy consumption and in polymerizing multifunctional monomers or monomers which undergo side reactions or are degraded under process conditions [35]. The enzymatic resolution of a substrate with a remote stereogenic center has been realized in the ﬁrst enantioselective synthesis of (S)monastrol [36]. An interesting high yield synthesis of 12-aminolauric acid from v-laurolactam has been developed by enzymatic transcrystallization using v-laurolactam hydrolase from Acidivorax sp. [37]. This method has been chosen because of low conversion ratios by the use of organic solvents and biphasic systems. Enzymatic
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transcrystallization starts with the addition of crystalline substrate to the aqueous reaction medium, which dissolves the substrate up to its solubility limit, and the enzymatic reaction can then give the soluble product, which will crystallize, when the product concentration from the enzymatic conversion exceeds the product solubility. Overall, the process resembles a SFPR system [11], where the crystalline substrate is converted into crystalline product in a highly efﬁcient and environmentfriendly process without organic solvent, acid or alkali. A nitrilase-catalyzed kinetic resolution of 2-cyano-1,4-benzodioxane and 2-cyano-6-formyl-1,4-benzodioxane to optically active 1,4-benzodioxane-2-carboxylic acids
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The formation and cleavage of carbon–carbon bonds is of prime importance for constructing the carbon skeleton not only in synthetic organic chemistry, but also in the
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metabolic pathways of living cells. Among the great variety of enzymes, hydroxynitrile lyases, aldolases, and transketolases have attracted much interest (Figure 5). Hydroxynitrile lyases have been valuable for manufacturing enantiopure target cyanohydrins from aldehydes, as versatile bifunctional building blocks for chemical synthesis [39]. Strategies for overcoming reaction limitations and suppression of nonenzymatic side reactions combine approaches from enzyme and reaction
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engineering [40]. Crude hydroxynitrile lyase has also been used for the enantioselective cyanohydrin synthesis in a microreactor [41]. Biocatalysis by means of aldolases offers a unique stereoselective and green tool to perform carbon–carbon bond formation or cleavage. Recent advances in aldolase-catalyzed stereoselective carbon– carbon bond formation reactions are valuable for generating molecular diversity and for synthetic improvements from small chiral polyfunctional molecules to highly
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complex oligosaccharide analogs [42]. Aldolase-catalyzed carbon–carbon bond formation has been used for the large-scale synthesis of a chloromethyl-substituted, a,b-unsaturated d-lactone [43]. The synthetic potential of thiamin diphosphate-dependent enzymes for asymmetric carboligations, such as asymmetric crossbenzoin condensations, has been extended appreciably and a variety of enantiomerically pure 2-hydroxyketones have been synthesized by enzymatic carbon–carbon
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Figure 5

Selected biocatalytic carbon–carbon bond formation reactions. The double aldol condensation of acetaldehyde with chloroacetaldehyde catalyzed by deoxyribose-5-phosphate aldolase (DERA-aldolase) has been applied industrially by DSM in the production of chiral lactones.

bond ligation of aldehydes [44]. The use of benzaldehyde lyase and benzoylformate decarboxylase in recombinant Escherichia coli resting cells in a MTBE/aqueous buffer biphasic medium has improved substrate solubility and extractive workup [45]. Another route to enantiomerically pure 2-hydroxy-ketones is the enzymatic chain elongation of aldehydes by a two-carbon unit, which can be catalyzed by transketolase and driven to completion by the use of the irreversible C2-ketol donor bhydroxypyruvate [46–48].
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A novel biocatalytic carbon–carbon bond formation reaction equivalent to Friedel–Crafts alkylation has been catalyzed by methyltransferases using S-adenosyl-Lmethionine and analogs [49]. A very broad range of acceptor substrates including cyclic and open-chain ketones as well as diketones and a-ketoesters and b-ketoesters have been found in the ﬁrst enzymatic asymmetric intermolecular aldehyde–ketone crosscoupling reaction, using the thiamine-dependent enzyme YerE [50]. Changing the substrate speciﬁcity of the
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Figure 6

Scheme of a biocatalytic multi-step and a biocatalytic multi-component reaction.

Industrial biocatalytic multi-step reactions
Multi-step processes coupling two or more biocatalytic reactions in one pot (Figure 6) are attractive because of the reduction in the number of process steps, productivity improvements and overcoming thermodynamic barriers [66]. One-pot synthetic methods involving multiple bond formation steps such as domino, tandem or cascade reactions eliminate also time-consuming recovery and puriﬁcation steps. Biocatalytic reactions have thereby been combined with other chemical or biocatalytic reactions. A prominent two-step example is the conversion of cephaCurrent Opinion in Biotechnology 2010, 21:713–724

losporin C to 7-aminocephalosporanic acid by D-amino acid oxidase and cephalosporin acylase. The synthesis of atorvastatin has been achieved by the biocatalytic reduction of ethyl-4-chloroacetoacetate using a ketoreductase-catalyzed reaction as the ﬁrst step and a halohydrin-dehalogenase-catalyzed substitution reaction of the chlorosubstituent with the cyano-group [52]. Another two-step reaction sequence has been used in the conversion of an aromatic alkene to a chiral 2-hydroxy ketone. The carbon– carbon double bond in the oleﬁn trans-anethole to para-anisaldehyde has been cleaved biocatalytically with a Trametes hirsuta extract and with molecular oxygen as oxidant. The second reaction step catalyzed the condensation of para-anisaldehyde to acetaldehyde by the enantiocomplementary C–C bond forming enzymes benzaldehydelyase and benzoylformatedecarboxylase, respectively, to yield either (R)-2-hydroxy-1-(4-methoxyphenyl)-propanone or (S)-2-hydroxy-1-(4-methoxyphenyl)-propanone [53].
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Natural product synthesis and modiﬁcation by biocatalytic multi-step reactions is of much interest because of the challenges in large-scale production of bioactive small molecules from natural sources or by total synthesis. Many opportunities exist for preparing a wide range of natural product variants due to the substrate ﬂexibility of the pathway enzymes. The bottom-up assembly of plant biosynthetic pathways in microorganisms is of interest for exploring the fascinating capabilities of the individual enzymes as well as for facilitating scalable production platforms for the synthesis of natural and unnatural alkaloids [54]. The optimization of biocatalytic pathways to macrotetrolides is highly attractive, because chemical synthesis of compounds like nonactin has not been competitive for large-scale production [55]. This is related to the exquisite selectivity and orthogonality of biocatalytic functional group transformations, which enables the organization of multi-step reactions in deﬁned reaction spaces in an analogous way as in biological cells, cell compartments or multi-enzyme machineries. The achievements of biocatalytic multi-step reactions serve as gold standard for the reaction development in organic chemistry [4].

developed for the high-yield synthesis of 3,4-dihydropyrimidine-2-(1H)-ones, consisting of the condensation of urea or thiourea with a substituted benzaldehyde and a 1,3-ketoester at room temperature in aqueous phosphate buffer pH 7.0 and using Saccharomyces cerevisiae as biocatalyst [59].

Development of new biocatalytic reaction methodology
Industrial applications aim at stable processes with robust, simple and sustainable operation and product recovery as well as high molecular economy [60–63]. The search for new biocatalytic reaction methodology is experiencing a boost by progress in a number of relevant areas like the connection between the broad set of natural product biosynthetic reactions and the genes that encode them [64] or the tremendous progress in engineering enzymes by directed evolution [65]. Whether the reaction is performed on a small or large scale, the conﬁnement or localization of enzymes in a certain reaction space, while retaining their catalytic activities under process conditions, is key. Expanding the organic chemistry of enzyme-catalyzed reactions and interfacing the enzyme reactions with classical chemical reactions in this reaction space, with no need for using protecting groups, is promising [66]. A concise approach to the synthesis of all 24 hexoses and 5-deoxy-hexoses, still ongoing, is based on a range of biocatalysts which interconvert polyols and ketoses, aldose isomerases for the equilibration of ketoses and aldoses, and D-tagatose-3epimerase for the C-3 equilibration of a wide range of substrates like ketoses, deoxysugars, and C-branched sugars [67]. An interesting biocatalytic domino reaction between phenol and various cyclic 1,3-dicarbonyl compounds yielded annulated benzofurans, using the enzymes tyrosinase and laccase from Agaricus bisporus [68]. The capturing and activation of carbon dioxide by enzymes has obtained increased interest [69], as on the one hand the chemistry of direct carboxylation reactions is underdeveloped and on the other hand many carboxylating and decarboxylating enzymes are occurring widely in nature. The novel continuous ﬂow enzymatic carboxylation of pyrrole to pyrrole-2-carboxylate by immobilized Bacillus megaterum represents an interesting green engineering approach [70]. Salicylic acid decarboxylase from Trichosporon monilliforme has been discovered to catalyze the enzymatic Kolbe–Schmitt reaction from phenol to salicylic acid [71]. 3,4-Dihydroxybenzoate decarboxylase from Enterobacter cloacae enabled the mild regioselective carboxylation of catechol to 3,4-dihydroxy-benzoic acid with 3 M potassium hydrogencarbonate at 308C [72].

Industrial biocatalytic multi-component reactions
While the tactics of step-by-step reactions is based on a cascade of subsequent functional group transformations, the goal for MCRs is to construct several bonds between the components by a parallel operation of different reactions with completely independent reactivity and selectivity. MCRs are therefore step-efﬁcient procedures converging towards the product and avoiding protecting group chemistry. MCRs enable building molecular complexity directly from more than two components. Although MCRs like the Strecker reaction are important industrial reactions in organic chemistry, the development of biocatalytic MCRs (Figure 6) has only recently attracted interest. A novel lipase-catalyzed direct Mannich reaction in water has been discovered, involving aniline, a nonenolizable substituted benzaldehyde as electrophile and the enolizable acetone as a source of nucleophile [56]. A sequence of a biocatalytic desymmetrization of a 3,4-substituted meso-pyrrolidin with monoamine oxidase N from Aspergillus niger and the use of the resulting enantiopure 1-pyrrolin as component in an Ugi-type 3-component reaction has been performed in two separate operations in order to achieve the best yields, diastereomeric ratio and ee values [57]. An interesting approach towards a biocatalytic asymmetric Strecker reaction has combined transimination with imine-cyanation in a double dynamic covalent system under thermodynamic control and subsequently coupled in a one-pot process with lipasecatalyzed transacylation under kinetic control [58]. A biocatalytic Biginelli 3-component reaction has been
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Conclusions
Biocatalytic single-step reaction platforms developed over the last years have progressed rapidly in the industrial
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production environment and many more methodologies developed at the research scale are waiting to be applied and to be scaled up. Discovery and development of novel biocatalytic single-step reactions continues to be important, especially in areas where no direct functional group transformation is known or where the known chemical transformation is lacking safety, selectivity or sustainability. The innate selectivity and orthogonality advantage of biocatalytic reactions bears a lot of potential for major improvements in multi-step reactions. Attention needs to be paid to both the molecular and the engineering aspects of the architecture of such biocatalytic multi-step systems. Whatever route is selected, key to further advances in sustainable chemical reactions is the development of novel biocatalytic reaction methodologies, which are modular, scalable, and compatible with the development of chemical reactions. The science, technology and industry of chemical synthesis and catalysis on the other hand is accepting established biocatalytic reaction platforms, because of the need for method and route simpliﬁcation, molecular economy, safety, health, and environment improvements. Therefore the knowledge building in industrial biocatalysis and its practical implementation is key for value creation in a future bioeconomy.